Colorimetric determinations of glycerol and of mono-, di-, and triglycerides (usually reported simply as "triglycerides") in biological fluids are carried out by various methods in many laboratories. The value of such determinations as an aid in diagnosis of atherosclerosis, diabetes mellitus, nephrosis and various other conditions is well established. See, Henry, Clinical Chemistry, Hoeber Division, Harper & Row, New York (1964), 864-870. The methods typically involve separation of triglycerides (as well as mono- and diglycerides) from other lipids such as phospholipids by extraction or chromatography. The separated triglycerides are then hydrolyzed to produce glycerol, which is then analyzed chemically, by chemical oxidation to formaldehyde and assay of the formaldehyde or by enzymatic determination. The enzymatic procedure uses the enzymes glycerol kinase ("GK", also referred to as "glycerokinase") glycerol-1-phosphate dehydrogenase (G-1-PDH) in the presence of adenosine-5-triphosphate (ATP) and oxidized nicotinamide adenine dinucleotide (NAD), according to the following scheme: ##EQU1## The reduction of NAD to NADH can be followed by ultraviolet spectroscopy. The glycerol determination can also be carried out by ultraviolet assay using glycerol dehydrogenase to catalyze the reaction:
Glycerol + NAD .fwdarw. NADH + dihydroxyacetone In the GK, G-1-PDH catalyzed procedure, hydrazine has usually been added to the reaction mixture to react with the dihydroxyacetone phosphate, to drive the reaction to the right. See, for example, Spinella et al., J. Lipid Research, 7, pp 167-169 (1966).
Methods have been described in which serum samples are hydrolyzed with alcoholic base and the neutralized extracts are assayed colorimetrically by coupling the GK, G-1-PDH, reactions to the diaphorase-catalyzed reaction of NADH with the tetrazolium dye, INT. Klotzsch et al., Abstract CC31, Technicon International Congress "Advances in Automated Analysis", Technicon Instruments Corporation, Tarrytown, N.Y. (1972). However, the previous alcoholic base hydrolysis procedures required saponification in heated ethanolic potassium hydroxide for long periods at elevated temperatures (e.g., thirty minutes at 55.degree.-70.degree. C.). See, Carlson et al., Clin. Chim. Acta, 4, 197-205 (1959). Such methods have thus been regarded as undesirably time-consuming and laborious. See, Stork et al., U.S. Pat. No. 3,759,793.